Organs in the vertebrate body are usually formed by large numbers of cells that are derived from multiple embryonic tissues and that have diverse physiological and structural functions. This complexity makes it difficult to unravel the networks that control the molecular mechanisms of organogenesis. In contrast, Kupffer's Vesicle (KV) is a'^highly organized embryonic structurethat is comprised of approximately four dozen cells. These cells are derived from Dorsal Forerunner Cells (DFCs) that migrate during gastrulation and then ingress into the tailbud to form the KV. Although the KV was described in 1868 and is highly conserved in teleosts, until our studies the function of KV was unknown. We found that KV epithelial lining has motile cilia that project into the lumen of KV and generate right-left asymmetric fluid flow. Using mutants, morpholino injections and laser ablations, we have discovered the function of the DFCs and KV: regulation of LR patterning in the brain, heart and gut primordia. We have shown that two T-box transcription factor genes, no tail (ntl) and spadetail (spt), have multiple roles in KV cilia gene expression, organogenesis, patterning and function. Some of these roles are distinct, and some are overlapping, suggesting that there are multiple cohorts of genes controlled by ntl, spt or combinatorial interactions between ntl, spt and other pathways. Our preliminary results indicate that the transcription factor genes rfx2 and hfh4 intersect with these T-box-dependent pathways; morphants of these genes do not alter KV formation, but alter the ability of cilia to function properly. The goal of this project is to elucidate the complex regulatory interactions, based on four transcription factor genes, ntl, spt, rfx2 and hfh4, that coordinate the formation, ciliagenesis and function of this simple but essential embryonic organ.